Impaired Responsiveness to the Platelet P2Y12 Receptor Antagonist Clopidogrel in Patients With Type 2 Diabetes and Coronary Artery Disease

JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY

VOL. 64, NO. 10, 2014

ª 2014 BY THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION PUBLISHED BY ELSEVIER INC.

ISSN 0735-1097/$36.00 http://dx.doi.org/10.1016/j.jacc.2014.06.1170

Impaired Responsiveness to the Platelet P2Y12 Receptor Antagonist Clopidogrel in Patients With Type 2 Diabetes and Coronary Artery Disease Dominick J. Angiolillo, MD, PHD,* Joseph A. Jakubowski, PHD,y José Luis Ferreiro, MD,* Antonio Tello-Montoliu, MD, PHD,* Fabiana Rollini, MD,* Francesco Franchi, MD,* Masafumi Ueno, MD,* Andrew Darlington, MD,* Bhaloo Desai, PHD,* Brian A. Moser, MS,y Atsuhiro Sugidachi, PHD,z Luis A. Guzman, MD,* Theodore A. Bass, MD*

ABSTRACT BACKGROUND Several studies have shown that patients with diabetes mellitus (DM) exhibit an impaired response to clopidogrel. This may contribute to their increased risk of recurrent atherothrombotic events, despite the use of dualantiplatelet therapy. The mechanisms for impaired clopidogrel response in DM patients have not been fully elucidated. OBJECTIVES The aim of this study was to explore the mechanisms for impaired clopidogrel-mediated platelet inhibition in patients with DM using a comprehensive methodological approach embracing both pharmacokinetic (PK) and pharmacodynamic (PD) assessments as well as ex vivo and in vitro investigations. METHODS Patients (DM, n ¼ 30; non-DM, n ¼ 30) with stable coronary artery disease taking aspirin 81 mg/day and P2Y12 antagonist naive were enrolled. Blood was collected before and at various times (0.5, 1, 2, 4, 6, and 24 h) after a 600-mg loading dose of clopidogrel. PD assessments included vasodilator-stimulated phosphoprotein, light transmission aggregometry, and VerifyNow P2Y12 ex vivo, before and after dosing and following in vitro incubation with escalating concentrations (1, 3, and 10 mM) of clopidogrel’s active metabolite (Clop-AM). Exposure to Clop-AM was also determined. RESULTS PD assessments consistently showed that during the overall 24-h study time course, residual platelet reactivity was higher in DM patients compared with non-DM patients. In vitro incubation with Clop-AM revealed altered functional status of the P2Y12 signaling pathway in DM platelets as measured by vasodilator-stimulated phosphoprotein, but not with other PD assays. Clop-AM exposure was w40% lower in DM patients than in non-DM patients. CONCLUSIONS The present study suggests that among DM patients, impaired P2Y12 inhibition mediated by clopidogrel is largely attributable to attenuation of clopidogrel’s PK profile. This is characterized by lower plasma levels of Clop-AM over the sampling time course in DM patients compared with non-DM patients and only modestly attributed to altered functional status of the P2Y12 signaling pathway. (J Am Coll Cardiol 2014;64:1005–14) © 2014 by the American College of Cardiology Foundation.

From the *University of Florida College of Medicine-Jacksonville, Jacksonville, Florida; yLilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana; and zDaiichi Sankyo Co., Ltd., Tokyo, Japan. This study was funded by an institutional grant from the University of Florida College of Medicine-Jacksonville and by a grant from The James and Esther King Biomedical Research Program (1KN01-33989) to Dr. Angiolillo. Daiichi Sankyo Co., Ltd., Tokyo, Japan provided clopidogrel active metabolite. Dr. Angiolillo has received consulting fees or honoraria from Bristol-Myers Squibb, Sanofi-Aventis, Eli Lilly, Daiichi Sankyo, The Medicines Company, AstraZeneca, Merck & Co., Evolva, Abbott Vascular, Bayer, and PLx Pharma; participates in review activities for Johnson & Johnson, St. Jude Medical, and Sunovion; and has received institutional grants from Bristol-Myers Squibb, Sanofi-Aventis, GlaxoSmithKline, Otsuka, Eli Lilly, Daiichi Sankyo, The Medicines Company, AstraZeneca, Evolva, and Gilead. Dr. Jakubowski and Mr. Moser are minor shareholders in Eli Lilly. Dr. Ferreiro has received honoraria for lectures from Eli Lilly, Daiichi Sankyo, and AstraZeneca. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Manuscript received February 11, 2014; revised manuscript received June 25, 2014, accepted June 25, 2014.

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D

ABBREVIATIONS AND ACRONYMS

ual-antiplatelet therapy with as-

METHODS

pirin and an antagonist of the P2Y 12 receptor is the cornerstone

PATIENT POPULATION. A total of 60 patients (30 DM

of treatment in patients with acute coronary

and 30 non-DM) with coronary artery disease (CAD)

syndrome (ACS) and in those undergoing

taking low-dose aspirin were prospectively recruited.

percutaneous coronary intervention (1–3).

Patients were screened at the outpatient clinic of the

Despite the development of newer antiplate-

Division of Cardiology–University of Florida College

let agents with more predictable pharmaco-

of Medicine Jacksonville. Patients were considered

concentration-time curve

dynamic (PD) response, clopidogrel is still

eligible for the study if they met all of the following

through the sampling time of

the most broadly used P2Y 12 receptor antago-

inclusion criteria: between 18 and 80 years of age, had

clopidogrel active metabolite

nist (4). However, clopidogrel is character-

angiographically documented CAD, on treatment

concentration

ized by a high interindividual variability in

with low-dose aspirin (81 mg/day) for at least 30 days

CAD = coronary artery disease

PD effects, and 5% to 44% of treated patients

as part of standard of care, and treatment naive

Clop-AM = clopidogrel’s active

exhibit impaired responses (5,6). Impor-

for P2Y 12 antagonism (ticlopidine, clopidogrel, pra-

metabolite

tantly, inadequate clopidogrel-induced anti-

sugrel, or ticagrelor) for at least 30 days. Patients

ACS = acute coronary syndrome(s)

ADP = adenosine diphosphate ANCOVA = analysis of covariance

AUC[0-tlast] = area under the

the last quantifiable

Cmax = maximal observed

platelet

an

were stratified according to DM status, defined ac-

plasma concentration

increased risk of recurrent ischemic events,

cording to World Health Organization criteria. All

CYP = cytochrome P-450

including stent thrombosis (5–9). Multiple

subjects with DM needed to be on treatment with oral

DM = diabetes mellitus

factors can contribute to variations in indi-

hypoglycemic agents and/or insulin for at least 2

LD = loading dose

vidual responses to clopidogrel (5,6,10,11).

months without any changes in their regimen (20).

LSM = least-square mean

Among these, PD investigations from our

Exclusion criteria were any of the following: use

LTA = light transmission

group and others have shown that patients

of any antiplatelet therapy other than aspirin in

aggregometry

with diabetes mellitus (DM) have impaired

the past 30 days, use of parenteral or oral anti-

MFI = mean fluorescence

clopidogrel-mediated

effects

coagulation in the past 30 days, active bleeding,

intensity

and higher rates of poor responsiveness

hemodynamic instability, any clinical indication to

PD = pharmacodynamic

than non-DM patients (12–16). This may

be on a P2Y12 receptor antagonist, hemoglobin A 1c

PGE1 = prostaglandin E1

contribute to the enhanced atherothrombotic

>12%, use of any drug interfering with cytochrome

PK = pharmacokinetic

risk that characterizes DM patients despite

P-450 (CYP) metabolism (fluconazole, ketoconazole,

PRI = platelet reactivity index

antiplatelet therapy (17).

voriconazole, etravirine, felbamate, fluoxetine, flu-

associated

with

antiplatelet

SEE PAGE 1015

PRU = P2Y12 reaction units VASP = vasodilator-stimulated

VASP-P = vasodilator-

is

voxamine, omeprazole, esomeprazole, lansoprazole,

PRP = platelet-rich plasma

phosphoprotein

effect

The mechanisms of impaired response to clopidogrel in DM patients remain to be fully

rabeprazole), cerebrovascular accident in the past 3 months, any active malignancy, platelet count <100  106/ ml, hemoglobin <10 g/dl, creatinine >2.5 mg/dl, liver disease (bilirubin levels >2 mg/dl), or pregnant

stimulated phosphoprotein

elucidated. A post-hoc subgroup analysis

and lactating females. The study complied with the

phosphorylation

suggested that the reduced ex vivo PD effects

Declaration of Helsinki, was approved by the Insti-

VN = VerifyNow

of clopidogrel among DM patients are sec-

tutional Review Board of the University of Florida

ondary to a less favorable pharmacokinetic

College of Medicine–Jacksonville, and all patients

(PK) profile (18). In particular, DM patients have

gave their written informed consent.

reduced plasma levels of clopidogrel’s active metabolite (Clop-AM) compared with those of non-DM pa-

STUDY DESIGN. This was a prospective study in

tients (18). An in vitro pilot study suggested that

which ex vivo and in vitro experiments were con-

platelets from DM patients are characterized by up-

ducted. Blood samples for analysis were collected by

regulation of the P2Y12 signaling pathway, which, in

the antecubital vein in anticoagulated tubes using a

turn, can reduce the PD effects of clopidogrel

19-gauge needle. The first 2 ml of blood sampled was

(19). Prospective studies specifically designed to

discarded to avoid spontaneous platelet activation. In

provide mechanistic insights of impaired clopidogrel

the ex vivo experimental component of our study,

response in DM are lacking. The aim of this pro-

eligible patients were administered a 600-mg loading

spective study was to explore the underlying mech-

dose (LD) of clopidogrel; blood samples for PK and PD

anisms of impaired clopidogrel-mediated platelet

assessments were collected at a total of 7 time points:

inhibition in DM patients using a comprehensive

baseline (before LD administration) and 30 min, 1 h,

methodological approach, embracing both PK and PD

2 h, 4 h, 6 h, and 24 h after LD. In the in vitro

assessments as well as ex vivo and in vitro

experimental design of our study, blood samples

investigations.

collected at baseline only (before LD administration)

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Impaired Response to Clopidogrel in Diabetes

1007

were used; PD testing was performed before and after incubation (for 30 min at 37  C) with escalating concentrations of Clop-AM (1, 3, and 10 m M). Daiichi

DM

Clopidogrel 600 mg

PD and PK analysis

Non-DM

Clopidogrel 600 mg

PD and PK analysis

Sankyo Co., Ltd. (Tokyo, Japan) provided Clop-AM. A flow diagram of the study design is presented in

Patients with CAD on aspirin 81 mg/day

Figure 1. PD ASSESSMENTS. Three different PD assays were

used for both the ex vivo and in vitro experimental designs, as described below. Vasodilator-stimulated

Baseline (time 0)

p h o s p h o p r o t e i n . The

30’ 1h 2h 4h

6h

24h

platelet reactivity index (PRI) was determined using In vitro escalating concentrations of Clop-AM

standard protocols for the vasodilator-stimulated phosphoprotein (VASP) assay (21–23). Briefly, VASP phosphorylation (VASP-P) was measured by quanti-

PD analysis

tative flow cytometry using commercially available labeled monoclonal antibodies (Biocytex Inc., Marseille, France). The mean fluorescence intensities

F I G U R E 1 Study Design

(MFIs) of VASP-P levels were measured after chal-

CAD ¼ coronary artery disease; Clop-AM ¼ clopidogrel active metabolite; DM ¼ diabetes

lenge with prostaglandin E 1 (PGE1 ) and PGE 1 þ aden-

mellitus; PD ¼ pharmacodynamic; PK ¼ pharmacokinetic.

osine diphosphate (ADP). PGE 1 increases VASP-P levels through stimulation of adenylate cyclase; ADP binding to purinergic receptors leads to inhibition of adenylate cyclase. Thus, the addition of ADP to PGE1-stimulated platelets reduces the levels of PGE 1 -

microbead agglutination assay with reagents specific for the pathways of interest. By combining ADP and

induced VASP-P. The PRI was calculated as follows:

PGE1 , the VN P2Y 12 assay measures platelet reactivity

[(MFI PGE 1)  (MFI PGE 1 þ ADP)/(MFI PGE1 )] $ 100%. A

changes that are relatively specific for P2Y12 antago-

reduced PRI indicates greater inhibition of the P2Y 12 signaling pathway (24).

nists. Optical signal changes are reported in P2Y12 reaction units (PRUs).

L i g h t t r a n s m i s s i o n a g g r e g o m e t r y . Light trans-

PK ASSESSMENTS. A commercial laboratory (Advion

mission aggregometry (LTA) was performed accord-

Biosciences, Inc., Ithaca, New York) blinded to

ing to standard protocols as previously described

the nature of the samples determined the plasma

(21–23). Briefly, blood was collected in sodium cit-

concentration of Clop-AM using liquid chromatog-

rate (3.8%) tubes. Platelet aggregation was then

raphy with tandem mass spectrometry, according to

assessed by the turbidimetric method in a 2-channel

standard protocols (26). Blood was drawn into stan-

aggregometer (Chrono-Log 490 Model, Chrono-Log

dard ethylenediamine tetraacetic acid tubes. Within

Corp., Havertown, Pennsylvania) using platelet-rich

30 s, a derivatizing agent (3 0 -methoxyphenacyl-

plasma (PRP). Platelet agonists included 5 and

bromide) was added to capture and stabilize the

20 m M ADP. PRP was obtained as a supernatant after

active metabolite. The geometric mean area under

centrifugation of citrated blood at 1,000 rpm for

the concentration-time curve through the sampling



10 min and was kept at 37 C before use. Platelet-poor

time of the last quantifiable Clop-AM concentration

plasma was obtained by a second centrifugation at

(AUC [0-tlast] )

2,800 rpm for 10 min. For each measurement, light

observed plasma concentration (Cmax) of Clop-AM

transmission was adjusted to 0% with PRP and to

was recorded.

100% with platelet-poor plasma. Curves were recor-

Sample size estimation and study endpoints.

ded for 6 min after ADP stimuli, and the level of

The sample size was determined on the basis of as-

platelet aggregation at 5 min was recorded, being

sumptions derived for the ex vivo component of our

more reflective of P2Y 12-mediated signaling (25).

experimental design, in particular, the comparison of

V e r i f y N o w P 2 Y 1 2 a s s a y . We used the VerifyNow

PRI values from patients with and without DM at 6 h

(VN) system (Accumetrics, San Diego, California), a

after administration of a 600-mg clopidogrel LD.

turbidimetric-based optical detection system that

Assuming a 13% SD and an w10%, dropout rate, we

measures platelet-induced aggregation as increased

would be able to detect a 10% difference in PRI with

light transmittance according to the manufacturer’s

60 patients (30 DM and 30 non-DM), with 95% power

instructions, as described (22,23). The VN system is a

and a 2-tailed alpha value of 0.05. PRI was chosen

was

calculated

and

the

maximum

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because it is the most specific marker for P2Y 12

with the Kolmogorov-Smirnov test. For baseline

receptor–mediated signaling (24,27). As part of our

characteristics, continuous variables are expressed as

experimental plan, we justified our study’s sample

mean  SD and categorical variables are expressed

size to detect differences in our ex vivo PD experi-

as frequency and percentage. The chi-square or

ments with established methods and then evaluated

Fisher exact test (if the expected value in any

the other PK and PD study components. There is a

cell was <5) was used to compare categorical vari-

paucity of published data, limiting the ability to

ables between 2 groups, whereas the Student t or

define a sample size. The endpoints of our study

Mann-Whitney U test was used to compare contin-

included PD assessments (24 h overall time course)

uous variables, where appropriate. An analysis of

measured by VASP-PRI, LTA, and VN-P2Y12 as part of

covariance (ANCOVA) method with a general linear

the ex vivo component of our experimental design;

model was used to evaluate all between-group

PK assessments (Clop-AM plasma concentrations,

comparisons, using the baseline value of the corre-

Cmax and AUC[0-tlast]) as part of the ex vivo component

sponding platelet function test and obesity (defined

of our experimental design; and PD assessments as

as a body mass index $30 kg/m 2) as covariates. A

listed in the preceding text as part of the in vitro

repeated-measures ANCOVA model, also adjusted

component of our experimental design.

by baseline platelet function value and obesity

STATISTICAL ANALYSIS. Conformity to the normal

status, was used to evaluate the overall difference

distribution was evaluated for continuous variables

between groups. ANCOVA models were performed for the previously mentioned analyses in line with other PK/PD studies (28,29); however, due to the small sample size (because of the PK/PD nature of

T A B L E 1 Baseline Characteristics of the Study Population

Variable

Age, yrs

the study), the dependent variable was not always normally distributed in every combination of values

DM (n ¼ 30)

Non-DM (n ¼ 30)

p Value

58.9  8.8

60.5  9.3

0.496

Male

19 (63.3)

21 (70.0)

0.584

Obesity, BMI $30 kg/m2

21 (70.0)

14 (46.7)

0.067

Race

0.786

of the covariate and levels of the factor. PD results are reported as least-square mean (LSM)  SE for the detailed analysis in the preceding text. A 2-tailed p value <0.05 was considered to indicate a

Caucasian

17 (56.7)

18 (60.0)

statistically significant difference for all analyses

African American

10 (33.3)

9 (30.0)

performed. Statistical analysis was performed using SPSS version 21.0 software (SPSS Inc., Chicago,

Hispanic

1 (3.3)

1 (3.3)

Other

2 (6.7)

2 (6.7)

28 (93.3)

26 (86.7)

0.671

Dyslipidemia

27 (93.1)

26 (86.7)

0.671

Active smoking

10 (33.3)

10 (33.3)

1.00

Previous MI

16 (53.3)

14 (46.7)

0.606

Hypertension

PAD

Illinois).

RESULTS

4 (13.3)

4 (13.3)

1.00

Previous PCI

17 (56.7)

15 (50.0)

0.597

Previous CABG

11 (36.7)

11 (36.7)

1.00

Previous stroke

6 (20.0)

1 (3.3)

0.103

LVEF, %

56.9  12.6

56.3  9.1

0.904

patients (DM, n ¼ 30 and non-DM, n ¼ 30) provided

Creatinine, g/dl

1.04  0.37

0.95  0.34

0.409

their written informed consent to participate and

Platelet count, 103/ml

211.5  69.7

244.0  56.2

0.076

completed the study. Baseline characteristics are

12 (40.0)

—

—

groups, except for obesity status, which was numerically higher in subjects with DM (p ¼ 0.067). HbA 1c

OAD

21 (70.0)

—

—

Aspirin

30 (100)

30 (100)

1.00

Beta-blockers

25 (83.3)

24 (80.0)

0.706

ACEIs/ARBs

21 (70.0)

22 (73.3)

0.940

Statins

23 (76.7)

25 (83.3)

0.905

CCB Nitrates

these declined to participate. Thus, a total of 60

summarized in Table 1 and were similar between

Medications Insulin therapy

PATIENT POPULATION. A total of 72 patients meet-

ing study inclusion criteria were identified; 12 of

8 (26.6)

8 (26.6)

1.00

10 (30.0)

13 (43.3)

0.712

levels were 7.45  1.13% in patients with DM. EX VIVO PD ASSESSMENTS. PRI levels (measured

by VASP) (Figure 2A) after a 600-mg clopidogrel LD were significantly higher in DM subjects compared with non-DM during the 24 h of crude analysis

Values are mean  SD or n (%).

(p ¼ 0.001) and after adjusting for baseline PRI

ACEIs ¼ angiotensin-converting enzyme inhibitors; ARBs ¼ angiotensin receptor blockers; BMI ¼ body mass index; CABG ¼ coronary artery bypass surgery; CCB ¼ calcium-channel blockers; DM ¼ diabetes mellitus; LVEF ¼ left ventricular ejection fraction; MI ¼ myocardial infarction; OAD ¼ oral antidiabetic drugs; PAD ¼ peripheral artery disease, PCI ¼ percutaneous coronary intervention.

values and obesity status (p ¼ 0.016). Platelet reactivity measured with LTA with ADP 20 mM after the 600-mg clopidogrel LD was significantly higher in DM subjects than non-DM subjects (p ¼ 0.005 in

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Impaired Response to Clopidogrel in Diabetes

the unadjusted analysis and p ¼ 0.022 after adjust(Figure 2B). LTA with 5 mM ADP showed higher platelet reactivity in patients with DM (p ¼ 0.032 in the unadjusted analysis and p ¼ 0.080 after adjusting for baseline platelet reactivity values and obesity) (data not shown). Parallel findings were observed with PRU values measured with VN-P2Y12 during the overall 24 h after the clopidogrel LD in the crude analysis (p ¼ 0.039 in the unadjusted analysis and p ¼ 0.056 after adjusting for baseline

A

course were lower in DM patients compared with non-DM patients (Figure 3). Overall exposure to Clop-AM and C max was significantly reduced in DM patients (Table 2). In particular, the geometric LSM AUC[0-tlast] in DM patients was 62.7% of that observed for non-DM patients (32.81 ng $ h/ml vs. 52.36 ng $ h/ml; adjusted p ¼ 0.02). Accordingly, the geometric LSM C max in patients with DM was 53.0% of that observed for patients without DM (19.77 ng/ml vs. 37.32 ng/ml;

90 70 60 50 40 30 20 10

levels measured by VASP than those from patients without DM, both in the unadjusted analysis (p ¼ 0.006) and after adjustment for baseline PRI and obesity (p ¼ 0.034) (Figure 4A). Furthermore, PRI after incubation with 1 m M (p ¼ 0.047) of Clop-AM was significantly higher in patients with DM, but did not

and obesity. LTA with 20 m M ADP showed a higher, but

24

100 90

Adjusted analysis: p=0.022

80 70 60 50 40 30 20 10 0 0 0.5 1

C

2

4

6

24

350 300

Adjusted analysis: p=0.056

250 200 150 100 50 0 0 0.5 1

reach statistical significance with 3 mM (p ¼ 0.254) and 10 m M (p ¼ 0.062) after adjustment for baseline PRI

6

Hours

P2Y12 Reaction Units

P2Y12 inhibition, reflected in significantly higher PRI

4

B

DM blood samples collected at baseline with escalating concentrations of Clop-AM showed attenuated

2

Hours

adjusted p ¼ 0.004). IN VITRO PD ASSESSMENTS. In vitro incubation of

Adjusted analysis: p=0.016

80

0 0.5 1

Platelet Aggregation - 20 µM ADP (%)

Clop-AM concentrations throughout the 24-h time

DM

0

PRU and obesity) (Figure 2C). PK ASSESSMENTS. After the 600-mg clopidogrel LD,

Non-DM

100

Platelet Reactivity Index (%)

ing for baseline platelet reactivity and obesity)

2

4

6

24

Hours F I G U R E 2 Ex Vivo Pharmacodynamic Assessments After

600-mg Loading Dose of Clopidogrel

nonsignificant, platelet aggregation in DM patients

(A) Platelet reactivity index measured by the vasodilator-

in the global analysis (p ¼ 0.068 in the unadjusted

stimulated phosphoprotein assay. (B) Platelet aggregation

analysis and p ¼ 0.228 after adjustment for baseline

measured by light transmission aggregometry after stimulation

platelet reactivity and obesity), and at each Clop-AM concentration, platelet reactivity did not signifi-

with 20 mM adenosine diphosphate (ADP). (C) P2Y12 reaction units measured by the VerifyNow-P2Y12 assay. Values are expressed as least-square means. Error bars indicate SE. The p values indicate

cantly differ between groups (Figure 4B). Similar

the overall differences between groups assessed by repeated-

trends were observed for LTA with 5 m M ADP

measures analysis of covariance and adjusted for baseline values

(data not shown). PRU values measured with VN-

and obesity. DM ¼ diabetes mellitus.

P2Y12 after in vitro incubation with escalating concentrations of Clop-AM were also similar between patients with and without DM, both in the global

DISCUSSION

analysis (p ¼ 0.389 in the unadjusted analysis and p ¼ 0.567 after adjustment for baseline PRU and

The results of the present study, which used a

obesity) and at each active metabolite concentration

comprehensive methodological approach embracing

(Figure 4C).

PK and PD assessments and ex vivo and in vitro

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T A B L E 2 PK Profile of Clopidogrel Active Metabolite in

non-DM

80

Clop-AM Concentration (ng/mL)

1010

DM

Diabetic and Nondiabetic Patients

Parameter

DM Geometric LS Means

Non-DM Geometric LS Means

Ratio of Geometric LS Means, %

AUC[0-tlast], ng$h/ml

32.81

52.36

62.7

0.02

Cmax, ng/ml

19.77

37.32

53.0

0.004

70 60 50

p value

40 Geometric least-square (LS) means and p values were calculated on logtransformed pharmacokinetic (PK) parameters using an analysis of covariance method with a general linear model and obesity status as a covariate. Geometric LS means are presented after back-transformation to the original scale.

30 20

AUC[0-tlast] ¼ area under the concentration-time curve through the sampling time of the last quantifiable clopidogrel active metabolite concentration; Cmax ¼ maximal observed plasma concentration; DM ¼ diabetes mellitus.

10 0 0 0.5 1

4

2

6

24

Hours

effects and higher rates of poor responsiveness

F I G U R E 3 Pharmacokinetics of Clop-AM After

600-mg Loading Dose

compared with non-DM patients (12–16). This may explain the characteristic enhanced ischemic risk in

Mean plasma levels of Clop-AM during the 24 h after a 600-mg loading dose of clopidogrel. Error bars indicate SE. Clop-AM ¼

DM patients, including high rates of stent thrombosis,

clopidogrel active metabolite; DM ¼ diabetes mellitus.

despite clopidogrel treatment (17). The mechanisms contributing to inadequate clopidogrel-induced antiplatelet effects in DM patients are likely multifactorial, although some may be specific to this patient

evaluations, platelet

P2Y 12

confirm

that

receptor

clopidogrel-mediated

blockade

responses

are

population, such as differences in plasma levels of procoagulant factors, oxidative stress, and cellular

impaired in patients with DM compared with non-DM

function (17,30,31). In particular, platelets from

patients. In particular, we found that residual

patients with type 2 DM have lost responsiveness to

platelet reactivity after treatment with clopidogrel is

insulin, leading to increased P2Y12-mediated sup-

higher in patients with DM than in non-DM patients,

pression of cyclic adenosine monophosphate and

as corroborated by multiple PD assays. In vitro incu-

decreased response to P2Y12 inhibitors (32–34). A

bation with escalating concentrations of Clop-AM

previous investigation by our group demonstrated

showed that, as measured by VASP, the functional

that DM platelets exposed in vitro to escalating con-

status of the P2Y 12 signaling pathway in platelets

centrations of Clop-AM had persistently higher PRI

from DM patients is significantly inhibited. In

levels than non-DM platelets, suggesting that P2Y12-

contrast, with other PD assays, only trends toward up-

mediated signaling is dysfunctional in DM patients

regulation of the P2Y 12 signaling pathway were

(19). Although the present results are consistent with

observed. Our study shows that Clop-AM exposure

these previous in vitro findings with VASP, no sig-

was w40% lower in DM patients than in non-DM

nificant differences were found with LTA and

patients. These findings suggest that overall im-

VN-P2Y12, which showed only numerical increases in

paired platelet P2Y 12 receptor blockade mediated by

platelet reactivity in DM patients compared with non-

clopidogrel may largely be attributable to abnormal-

DM patients. These findings may also be due to the

ities in the Clop-AM PK profile (“drug exposure”) and

greater specificity of VASP-PRI for the P2Y 12 signaling

only to a much lesser degree to platelet dysfunction

pathway (24,27).

(drug response) (Central Illustration).

Inadequate clopidogrel-induced platelet inhibition

Several studies have demonstrated the clinical

among DM patients has also been attributed to sup-

benefit associated with platelet P2Y 12 receptor inhi-

pressed Clop-AM circulating levels. Erlinge et al. (18)

bition by clopidogrel, particularly in high-risk set-

conducted a post-hoc analysis of a small DM sub-

tings (1–3). However, a significant number of patients

group (n ¼ 9) derived from a prospective PK/PD

continue to experience poor response to clopidogrel

investigation and showed that DM patients were

and are at increased risk of adverse outcomes (4–9).

overrepresented among poor responders identified in

Multiple factors can contribute to these findings

a prospective evaluation comparing prasugrel and

(5–11). Among these, PD investigations (from our

clopidogrel and had significantly lower Clop-AM

group and others) have shown that patients with

levels. However, this study did not find any differ-

DM have impaired clopidogrel-induced antiplatelet

ences between DM and non-DM patients in either PD

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Impaired Response to Clopidogrel in Diabetes

effects after oral administration of clopidogrel or concentration (10 m M) of Clop-AM. Furthermore, only a trend toward a lower Clop-AM AUC among DM patients was observed (18). Overall, these findings were likely due to the small size of this analysis. Our investigation expands on this previous post-hoc analysis with our prospective, comprehensive PK and PD assessments and ex vivo and in vitro experiments using an array of assays in a larger study population. We showed that Cmax and the AUC of

A

findings suggest that impaired platelet P2Y 12 receptor blockade following clopidogrel is largely attributed to abnormalities in clopidogrel’s PK profile and only in “small” part attributed to platelet dysfunction. The mechanisms leading to abnormalities in clopidogrel’s PK profile among patients with DM are not fully understood and likely imply multiple contributing factors. Indeed, it is well-known that DM patients are affected by gastrointestinal and

p=0.047

70

p=0.254

60 50 40

p=0.062

30 20

Adjusted analysis: p=0.034

10 Baseline

90 80

p=0.316

60 50 40 30 20

p=0.433

Adjusted analysis: p=0.228

0

Baseline

hydrolysis. The need for more effective platelet-inhibiting strategies is underscored by the findings of the present investigation and the established knowledge that

P2Y12 Reaction Units

carboxylic acid metabolite; 3) reduced

3µM

10µM

C 350

hepatic CYP activity; and 4) increased Clop-AM

1µM

Concentrations of Clop-AM

300

inactive

p=0.194

10

among patients with DM may include: 1) decreased 2) increased clopidogrel prodrug hydrolysis to an

10µM

p=0.214

70

leading to abnormalities in clopidogrel’s PK profile gastrointestinal absorption of clopidogrel prodrug;

3µM

100

hepatic abnormalities that can affect drug absorption and metabolism (35–37). Therefore, factors

1µM

Concentrations of Clop-AM

B Platelet Aggregation - 20 µM ADP (%)

trations for the in vitro tests. Overall, our study

90

DM

p=0.070

80

0

Clop-AM were lower in DM patients than in non-DM patients. We also used a range of Clop-AM concen-

Non-DM

100

Platelet Reactivity Index (%)

in vitro PD effects after incubation with a single high

p=0.774

p=0.168

250

p=0.599

200 150 100 50 0

p=0.997

Adjusted analysis: p=0.567

Baseline

1µM

3µM

10µM

Concentrations of Clop-AM

patients with DM remain at risk of ischemic recurrences (17). To achieve this goal, several pilot PD

F I G U R E 4 In Vitro PD Assessments After Incubation With

studies have been conducted specifically in DM

Escalating Concentrations of Clop-AM

patients (21–23,38). Indeed, among currently available strategies, use of the novel and more potent P2Y12 receptor inhibitors prasugrel and ticagrelor are

(A) Platelet reactivity index measured by the vasodilator stimulated phosphoprotein assay. (B) Platelet aggregation measured by light transmission aggregometry after stimulation with 20 mM

the most promising. Recently, Alexopoulos et al. (39)

adenosine diphosphate. (C) P2Y12 reaction units measured by the

showed that DM patients with ACS undergoing

VerifyNow-P2Y12 assay. Values are expressed as least-square

percutaneous coronary intervention and pretreated

means. Error bars indicate SE. The p values indicate the overall

with clopidogrel achieved higher inhibition when switched to ticagrelor compared to when switched

differences between groups assessed by repeated-measures analysis of covariance and adjusted for baseline values and obesity. Abbreviations as in Figure 1.

to prasugrel. Although prasugrel achieves potent PD effects, largely attributed to its more favorable PK

profile

(i.e.,

ability

to

generate

its

active

metabolite) than that of clopidogrel, DM status also

whether DM status modulates plasma levels of

affects plasma levels of prasugrel active metabolite

ticagrelor and its CYP3A4-derived metabolite (AR-

(18,28).

of

C124910XX). Furthermore, it is possible that because

all oral P2Y12 receptor inhibitors or just of thieno-

ticagrelor is administered twice daily, which may be

pyridines remains to be established, as it is unknown

more optimal for patients with high platelet turnover

Whether

this

is

a

characteristic

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SEPTEMBER 9, 2014:1005–14

have a greater net clinical benefit in DM patients (41,42). STUDY LIMITATIONS. Our study demonstrates that im-

paired clopidogrel-mediated P2Y 12 inhibition among DM patients is mainly caused by lower Clop-AM exposure. However, we were not able to determine whether this reflects impaired absorption or metabolism or both (35–37). Plasma levels of clopidogrel prodrug would have helped to differentiate between these possibilities. Nevertheless, this does not affect the conclusions of our study. Because our investigation was performed in patients with stable CAD, these findings require confirmation in the setting of DM patients with ACS who are characterized by a hyperreactive platelet phenotype and are more susceptible to absorption and metabolism abnormalities (43,44). However, in the ACS setting, the novel P2Y12 receptor antagonists prasugrel and ticagrelor are now more frequently used, and clopidogrel remains the standard of care for stable CAD patients, the target population of the present investigation (4). Although this is among the most comprehensive explorations of the mechanisms associated with differences in clopidogrel response profiles between DM and non-DM platelets, the complex nature of the experiments limited our study to a relatively small number of patients. Indeed, the inherent differences between DM and non-DM patients indicate

that

additional

confounders,

others

than

those already accounted for in our statistical adjustments, may emerge in a larger study. Finally, C E N T R A L I L L U S T R A T I O N Mechanism of Impaired Clopidogrel-Induced P2Y 12

Receptor Blockade in Patients With DM

we did not genotype for CYP2C19 polymorphisms associated with differences in PK/PD profiles in

Adenosine diphosphate (ADP) (blue squares)–driven platelet activation and aggregation

our study; thus, we cannot exclude an allelic fre-

contribute to atherothrombotic events. Clopidogrel (red dots), after conversion to its

quency imbalance between the DM and non-DM

active metabolite (green triangles), antagonizes P2Y12, the platelet ADP receptor. In

cohorts (10,11).

diabetes mellitus (DM) patients treated with clopidogrel, impaired platelet P2Y12 blockade is largely attributed to marked reductions (w40% lower than nondiabetic subjects) in the pharmacokinetic profile of clopidogrel’s active metabolite (drug exposure) and attributed to a much lesser degree to altered functional status of the P2Y12 signaling pathway (drug response).

CONCLUSIONS Patients with DM exhibit an impaired platelet inhibitory response to the P2Y 12 receptor inhibitor clopidogrel. The present mechanistic study suggests

rates such as those with DM, it may lead to more

that this can largely be attributed to abnormalities in

consistent levels of platelet inhibition (40). How-

clopidogrel’s PK profile, characterized by reduced

ever, the rate of high platelet reactivity was ex-

plasma levels of Clop-AM that lead to reduced PD

tremely low without a significant difference between

effects, and can only be attributed to a much lesser

prasugrel and ticagrelor (39). These findings may

degree to dysfunctional P2Y12 signaling pathway

explain why both prasugrel and ticagrelor were

status.

beneficial in the DM cohorts of the TRITON-TIMI 38 (Trial to Assess Improvement in Therapeutic

REPRINT REQUESTS AND CORRESPONDENCE: Dr.

Outcomes by Optimizing Platelet Inhibition with

Dominick J. Angiolillo, Division of Cardiology, Uni-

Prasugrel-Thrombolysis In Myocardial Infarction 38)

versity of Florida College of Medicine–Jacksonville,

and PLATO (Platelet Inhibition and Patient Outcomes)

655 West 8th Street, Jacksonville, Florida 32209.

trials, respectively, although prasugrel appeared to

E-mail: [email protected]fl.edu.

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SEPTEMBER 9, 2014:1005–14

Impaired Response to Clopidogrel in Diabetes

PERSPECTIVES COMPETENCY IN MEDICAL KNOWLEDGE: Impaired

TRANSLATIONAL OUTLOOK: Development of

responsiveness to the P2Y12 receptor inhibitor clopidogrel

methods to enhance the pharmacokinetic profile of

contributes to the increased risk of recurrent athero-

clopidogrel and increase drug exposure in patients with

thrombotic events in patients with diabetes mellitus.

diabetes mellitus will likely prove more effective than interventions that alter the P2Y12 signaling pathway in an effort to improve platelet inhibition in response to the drug.

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KEY WORDS coronary disease, diabetes mellitus, platelets